Multidrug-resistant Escherichia coli causing canine pyometra and urinary tract infections are genetically related but distinct from those causing prostatic abscesses

Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.


Phylogroups of E. coli isolated from pyometra, prostatic abscesses, and bloodstream infections
Out of 104 E. coli isolates, 101 isolates were recovered from dogs suffering from canine pyometra (61 isolates), PAs (38 isolates), and BSIs (2 isolates), and the other three isolates were recovered from cats with BSIs.

Virulence gene carriage
A total of 104 E. coli isolates recovered from pyometra, PAs, and BSIs were screened for VG carriage.The isolates that carried ≥ 2 out of 5 VGs (papC, sfa/foc, afa, iutA, and kpsII) were categorised as ExPEC, whereas those carried ≥ 3 out of 4 VGs (yfcV, vat, fyuA, and chuA) were classified as UPEC 4,6 .Table 2 presents the proportions of E. coli isolated from pyometra, PAs, and BSIs, classified into UPEC and ExPEC pathotypes.A significantly higher proportion of pyometra strains was categorised as UPEC compared to PA.However, proportions of E. coli classified as ExPEC were not significantly different.A range of 3-22 VGs were detected in the E. coli isolates.VGs associated with UPEC (yfcV, fyuA, chuA), as well as irp1, hlyE, and kpsII, were significantly more prevalent in isolates from pyometra than in PAs.ExPEC VGs, including papC, iha, afa, hlyA, and cnf, were common in isolates from pyometra and PAs but were not detected in BSI isolates.VGs were associated with the phylogroups in which high numbers of VGs, including sfa, papC, yfcV, tsh, hlyA, hlyE, vat, fyuA, irp1, chuA, and kpsII, were significantly presented in phylogroup B2.

Genome characteristics of E. coli isolates subjected for WGS analysis
The genome of 12 E. coli strains, isolated from pyometra (4 isolates), PAs (5 isolates), and BSIs (3 isolates), as indicated in Supplementary Fig. S2, were sequenced and compared to the genome of seven E. coli strains from UTIs.Chromosome sizes ranged from 4,836,704 to 5,583,986 bp and had a G + C content from 50.42 to 50.86%.

Clonal relationship of ExPEC strains from different disease patterns
Three pyometra strains and two BSI strains shared common genetic background of high-risk E. coli, including ST131-B2 (ST-phylogroup), ST1193-B2, and ST648-F, with intra-clonal variation.The core genome multilocus sequence typing (cgMLST) analysis revealed only 18 and 39 different loci between the ST1193-B2 strains from BSI and UTI, respectively, compared with the pyometra strain.ST648-F strains from pyometra and UTI were more closely related, as evidenced by a difference of only 182-189 loci, while these strains were distant from the ST648-F strain from BSI with more than 975 loci difference.Up to 686 different loci were observed between ST131-B2 strains from pyometra and UTI. Figure 1 illustrates the minimum spanning tree based on cgMLST of the 19 E. coli isolates, representing clonal relationships among and within the high-risk clones.
The single-nucleotide polymorphisms (SNP) -based phylogenetic tree, based on 2,438 core genes, clustered the E. coli strains into four clades (Fig. 2).Phylogroup B2 strains were specific to the closely related clade 1 and 2, while phylogroup F was specific to clade 3. Phylogroup A, B1 and D were separately grouped in clade 4. In addition to 2438 core genes, accessory genes, comprising 8950 cloud genes and 5000 shell genes, were found among this E. coli population.Based on the presence and absence of the accessory genes, three clusters specifically grouped the phylogroup B2, F and a group of phylogroup A, B1 and D. Additionally, a strong correlation was observed between the accessory genome and the coreSNP analysis (see Supplementary Fig. S3 online).

Pathogenicity islands (PAIs) and plasmids
PAI(s) were identified on the chromosome of 16 E. coli isolates (Fig. 3), and they were absent in three isolates, including ST44-A and ST744-A from PAs, and ST648-F from BSI.The phylogroup B2 isolates contained up to five PAIs that carried several VGs (Fig. 3 and Supplementary Table S3).PAIs that co-harbored iss and ompT were specifically found in phylogroup A and B1.Common yersiniabactin-associated PAIs, classified as high PAIs (HPI), were present across multiple phylogroups, except for phylogroup A. However, a variant of the yersiniabactin-associated PAIs containing the type IV secretion system (T4SS) was detected in CUVET16-242 of phylogroup A from UTI.Moreover, a PAI containing sfa/foc genes was unusually inserted following pheU, also found in strain CUVET20-PYO5 (phylogroup B2), which carried the most numerous VGs.The aerobactinencoding gene cluster was abundantly found on multi-replicon plasmids that carried ARGs, considered hybrid plasmids.The numbers and replicon types of resistance and virulence plasmids are illustrated in Fig. 2. Details of total plasmids and ARG and VG localisation are provided in Supplementary Table S2.

Virulome and resistome
Among the 19 sequenced E. coli isolates, 139 VGs encoding 27 virulence factors, and 41 ARGs mediating resistance to 10 antimicrobial classes were identified (Fig. 2 and Supplementary Fig. S4).Overall, high numbers of VGs were observed in phylogroup B2 (78-97 gene, with an average of 85.3 genes), followed by phylogroup F (63-95 genes, with an average of 76.4 genes).PAI-encoded ExPEC toxin and capsular genes, including cnf1, hlyA, and kps, were specifically present in phylogroup B2 and F isolates.In contrast, VG carriage was fewer in the strains in phylogroup A (35-60 genes, with an average of 51.2 genes) and phylogroup B1 (38-56 VGs with an average of 47 genes).Strain CUVET21-PYO5, belonging to ST998-B2, carried 97 VGs encoding 22 virulence factors but harbored only two ARGs, including bla CTX-M-14 and mph(A) on the chromosome.Strain CUVET19-1426, belonging to ST13037-A isolated from PA, had the lowest VG carriage, containing only 35 genes, but carried the highest number of ARGs on plasmids.
Regarding ARG carriage, phylogroup A isolates carried the highest number (11-17 genes with an average of 13.2 genes), while phylogroup B2 isolates had the lowest number (2-16 genes with an average 8.3 genes).Localisation of ARGs on the chromosome and plasmids in each isolate is indicated in Fig. 2. The majority of ARGs were located on plasmids in all plasmid-carrying isolates, except for ST12-B2.Larger than 120 kb hybrid virulence/MDR multi-replicon plasmids, carrying aerobactin genes, were more common in the phylogroup A and F isolates (Fig. 2 and Supplementary Table S2).Among the 14 bla CTX-M -positive isolates, six strains harbored bla CTX-M on the chromosome, four of which were phylogroup B2.The remaining eight bla CTX-M -positive www.nature.com/scientificreports/isolates contained the gene on IncF plasmids, except for one strain carrying on an IncR/N plasmid.Only within ST648, chromosomal bla CMY-2 and plasmidic bla CMY-148 were detected.Among the 19 E. coli strains, mutation at gyrA:pS83L was detected in 18 strains, but 17 expressed FQ R .Three mutations of quinolone resistancedetermining regions (QRDR), including gyrA:pS83L, gyrA:pD87N, and parC:pS80I, were observed in 15 FQ R strains.Of these, six strains harbored plasmid-mediated quinolone resistance (PMQR) genes, distributed on the chromosome and plasmids.Despite the absence of mutation of QRDR, one strain in phylogroup B2 contained two PMQR genes mediating FQ R , including aac(6')-Ib-cr on chromosome and qnrB6 on a plasmid.

Discussion
In addition to UTIs, E. coli phylogroup B2 was associated with pyometra, PAs, and BSIs and contained the most abundant VGs.The majority of E. coli isolated from pyometra, PAs, and BSIs in this study belonged to phylogroup B2, as those isolated from extraintestinal infections in pets and humans in previous studies 8, [13][14][15][16] .However, the characteristics of BSI isolates should be interpreted with consideration regarding the small number of isolates, which might lead to bias in statistical analysis.The virulence genotyping revealed that 72.1% and 80.3% of the E. coli isolated from pyometra contained VGs of ExPEC and UPEC pathotypes 5,6 , respectively, which were likely associated with the phylogroup rather than the disease patterns.Due to the lower numbers of phylogroup B2 in PA strains, ExPEC and UPEC genotypes were infrequently found in E. coli isolates from PAs. Non-B2 phylogroups isolated at lower frequencies in all diseases and contained less complete VGs required for the steps of ExPEC pathogenesis.In other extraintestinal infections such as UTIs, non-B2 E. coli phylogroups contained fewer VGs; however, genes encoding adhesins and siderophores were detected in most of the isolates 4 .The binding of type 1 fimbriae encoded by fimA to uroepithelium and uteroepithelium is considered a critical step in adhesion during uropathogenesis and uteropathogenesis, respectively 17,18 .The presence of specific ExPEC adhesin genes, such as sfa encoding S-fimbriae, can support successful extraintestinal colonisation.S-fimbriae specifically bind α-sialyl-2-3-β-galactose (NeuAc-α 2,3-Gal) on the animal cell surface and can enhance adhesion of UPEC 17 .The expression of fyuA and irp1 (yersiniabactin) in alkaline conditions, like urinary bladder and uterus, assists in iron acquisition for extraintestinal survival 6,14,19,20 .Likewise, hemolysin A and capsular genes are more specific to phylogroup B2 in UPEC and NMEC strains 13 .Therefore, the extraintestinal infections frequently caused by E. coli phylogroup B2 could be enhanced by all VGs corresponding to adhesion, toxin production, iron acquisition, and immune evasion.However, adhesins and iron acquisition systems were likely required in the extraintestinal pathogenesis of any E. coli phylogroups.
According to PAI and virulome analyses, PAIs served as the key genetic determinants for the virulence of the ExPEC isolates.High numbers of VGs, such as capsular, hemolysin A, and pyelonephritis-associated pili genes,    were consistently detected on the PAIs found in phylogroup B2 across various disease patterns.Essentially, HPI containing yersiniabactin gene cluster, were non-specifically widespread in all phylogroups, supporting the fundamental requirement of an iron-acquiring system to survive outside the intestine 21 .Evidence supports the notion that pathogenic attenuation of E. coli strains containing the HPI can be caused by irp2 inactivation 22 .In phylogroup B2, the pheV-PAI containing iron-regulated gene homologue adhesin (iha), secreted autotransporter toxin (sat), aerobactin (iuc and iut), and capsular (kps) genes, were conserved in isolates from any infections.Additionally, the pheU-PAI containing the pap gene cluster, hemolysin A (hlyA), cytotoxin necrotising factor (cnf), and contact-dependent growth inhibitor (cdi), were more specific to ST131-B2.These PAIs bearing VGs associated with uropathogenesis are detected in phylogroup F and D from urinary sources 23 , but not in these phylogroups isolated from PAs, and BSIs in our study.Sharing of ExPEC VGs among the E. coli clones could be described by the presence of S fimbriae-associated PAI (pheU-PAI) in ST998-B2 and ST617-A.Additionally, the PAI-encoded aerobactin genes (iutA and iuc gene cluster) in B2 strains were carried on plasmids in non-B2 strains.The acquisition of MGEs encoding ExPEC VGs as part of the accessory genome, such as PAI-encoded sfa/foc and plasmid-encoded iut/iuc, could be considered an evolutionary process leading to ExPEC, as assessed by the ExPEC criteria 6 .
The genomic background of MDR ExPEC from pyometra and BSIs using multilocus sequence typing (MLST) analysis showed a clonal relationship to high-risk ExPEC strains causing UTIs, including ST131-B2, ST1193-B2, and ST648-F, which are important for global monitoring, but PA isolates appeared to be distinct.Canine uterosepsis caused by E. coli ST1193-B2 was presented in a dog concurrently suffering from pyometra and BSI.Due to the identical genetic characteristics of strains from the uterus and blood, only the strain isolated from blood (CUVET21-H2) was subjected to WGS, revealing significant VG carriage such as the capsule production gene kpsII for immune evasion.E. coli ST1193 contains VGs similar to those found in ST131 but commonly carries the senB gene on IncF plasmids and is associated with infections in humans in the community rather than nosocomial infections 24 .
Intra-clonal variation was observed in the high-risk STs by coreSNP analysis, cgMLST, variations in O and H antigens, as well as differences in the type and location of accessory genes associated with virulence and resistance.E. coli ST131-B2 strains from both pyometra and UTI exhibited the greatest genetic diversity within the same ST.The chromosomally encoded bla CTX-M-15 E. coli ST131 strain associated with UTI belonged to clade C2/H30Rx, which is related to the predominant bacteremic E. coli ST131 SEA-C2 clone in Southeast Asia and disseminated worldwide 25 .Furthermore, heterogeneity was observed among ST1193 strains in terms of 3GC R development, illustrating acquisition of bla CTX-M , which was found in one strain from UTI.The emergence of MDR E. coli ST1193 has been increasingly reported, representing a successful global high-risk clone following the footsteps of E. coli ST131 26 .In Thailand, ST131, ST648, and ST1193 are the most common STs associated with high-risk ESBL-producing E. coli carried by hospitalised patients 27 .High-risk MDR E. coli clones dominate the population of 3GC R and FQ R E. coli.Selecting 3GC R and FQ R isolates in AMR monitoring of ExPEC in animals could support the findings of high-risk clones of global concern.
Although a low number of 3GC R was observed, the carriage of bla CTX-M was found to be common in 3GC R ExPEC associated with high-risk E. coli clones from UTIs, and pyometra, as well as in non-high-risk clones from PAs.The detected bla CTX-M variants in this study, including bla CTX-M-15, bla CTX-M-14 , bla CTX-M-27 , and bla CTX-M-55 , are the most common variants in E. coli across humans, livestock, and the environment 28 , suggesting the wide dissemination within the E. coli population.The predominant bla CTX-M variants were the same as those found in canine and feline UPEC in Thailand 12 .The presence of bla CTX-M genes on plasmids is more common; however, chromosomal integration of bla CTX-M and AmpC, mediated by ISEcp1, supported the stabilisation of the gene in the genome.This could facilitate clonal dissemination, as found in ST131 29,30 .Mutations in the QRDR region in gyrA and/or parC are the major mechanism of FQ R in bacteria and a primary factor for the successful clonal spread of ExPEC ST131 and ST1193, contributing to their emergence worldwide 26 .PMQR genes are an alternative mechanism in enterobacteria, developed through the acquisition of MGEs.In this study, most of the ARGs were carried by multi-replicon IncF plasmids, which plays a crucial role in bacterial evolution and primarily influences resistance evolution in these ExPEC strains 31 .The multiple recombination of the IncF plasmids, generating large multi-replicon plasmids containing numerous ARGs, VGs, and transfer modules, results in greater stability within bacterial hosts and more efficient transfer 32,33 .The evolution from IncF plasmid carriage and plasmid recombination might potentially promote E. coli to persist outside the gastrointestinal tract through iron acquisition in infections and enhance resistance to antimicrobials during the treatment.
Pan-genome analysis demonstrates the concurrent evolution of core genes by SNPs and accessory genes by gene acquisition in each lineage, indicating co-evolution in both core and accessory genomes.There is a high correlation between the core genome and accessory genome of E. coli ST117, reflecting diverse evolution in this clone by acquisition of PAIs and ARGs 34 .Overall, both core and accessory genome of the ExPEC strains are associated with evolution within the lineages and AMR.However, limitations of the study were addressed by the low numbers of WGS-sequenced isolates that were selected based on the highest numbers of virulence and resistance genes among the isolates from different disease patterns, as well as the far fewer isolates from BSIs.
This study highlighted the virulence traits of ExPEC causing canine pyometra and PAs, and BSIs in dogs and cats, most of them belong to phylogroup B2 containing numerous ExPEC VGs similar to those found in UPEC.However, non-B2 phylogroups may at least require adhesion and an iron-acquiring system.The 3GC R and FQ R E. coli containing high numbers of ARGs and VGs from pyometra and BSIs are related to three high-risk E. coli lineages: B2-ST131, B2-ST1193, and F-ST648.Genetic features of E. coli from PAs are distinct from those of other extraintestinal infections.Virulence in the lineages is presented by PAIs in the chromosome, and IncF plasmids, especially multi-replicon plasmids, also play a crucial role in MDR evolution.Thus, pets act as a reservoir of high-risk E. coli, posing a risk of transmission to humans in the community.Effective diagnostic antimicrobial susceptibility testing to improve antimicrobial uses should be encouraged for the treatment of diseases caused

Ethics approval
This study was conducted according to the faculty regulations and approved by the Institutional Biosafety Committee of Faculty of Veterinary Science, Chulalongkorn University (CU-VET-IBC) (Protocol code IBC 2131030) on 13 December 2020.The authors confirm their adherence to the ethical policies outlined in the journal's author guidelines.All uterine samples utilised for bacterial isolation in this study were obtained from canine patients who had undergone ovariohysterectomy for therapeutic purposes, under the care of licensed veterinarians in small animal hospitals, with the consent of the owners.The acquisition of samples from animal patients was conducted in compliance with the approval granted by the Institutional Animal Care and Use Committee of the Faculty of Veterinary Science, Chulalongkorn University (Protocol code 2131054) on January 26, 2021.

Bacterial isolates
A total of 104 E. coli from non-urinary extraintestinal infections, including canine pyometra (n = 61), canine PAs (n = 38), and canine and feline BSIs (n = 5) collected from 2016 to 2021, were included for E. coli phylogrouping, ExPEC VG detection, and screening of FQ R and 3GC R .The 61 E. coli strains from pyometra were isolated from the endometrium of 48 out of 100 uteri of female dogs that underwent ovariohysterectomy for surgical treatment of canine pyometra at the Obstetrics, Gynecology, and Reproduction Unit, Small Animal Teaching Hospital, Chulalongkorn University, Thailand.From 12 dogs, two distinct E. coli isolates were recovered from the uterus, each presenting different colony characteristics.Additionally, two separate E. coli isolates were recovered: one from the endometrium and another from the blood of the same dog suffering from pyometra, which had progressed to sepsis.The 38 isolates from canine PAs were obtained from PA exudate collected by ultrasoundguided aspiration, and the 5 isolates (2 from dogs and 3 from cats) causing BSI were isolated from positive blood culture bottles using the BD BACTEC™ FX blood culture system (Becton-Dickenson, USA).Isolates that tested positive for ≥ 2 out of 5 VGs (papC, sfa/foc, afa, iutA, and kpsII) were presumably classified as ExPEC, whereas isolates demonstrating ≥ 3 out of 4 VGs (yfcV, vat, fyuA, and chuA) were presumably classified as UPEC 4,6 .

Screening of FQ-and/or 3GC-resistant E. coli and antimicrobial susceptibility testing
FQ R and 3GC R were examined in all 104 E. coli isolates using ciprofloxacin, cefotaxime, and ceftazidime disk diffusion methods 36 .Isolates displaying resistance to cefotaxime and/or ceftazidime were included for detection of ESBL and AmpC β-lactamase production by the combination disk test and cefoxitin disk diffusion test, respectively.The AMR phenotypes of FQ R and/or 3GC R E. coli were determined using broth microdilution with a customised Sensititre™ plate model COMPGN1F (Thermo Scientific, UK).The plate contained amikacin, amoxicillin/clavulanic acid, ampicillin, cefazolin, cefovecin, cefpodoxime, ceftazidime, cephalexin, chloramphenicol, doxycycline, enrofloxacin, gentamicin, imipenem, marbofloxacin, orbifloxacin, piperacillin/tazobactam, pradofloxacin, tetracycline, and sulfamethoxazole/trimethoprim.Resistance to the drugs was interpreted according to the minimum inhibitory concentration (MIC) breakpoints for veterinary isolates from the Clinical and Laboratory Standards Institute 37 .

Strain selection for whole genome sequencing
The clonal relatedness of 44 FQ R and/or 3GC R E. coli from pyometra (19 isolates), PAs (20 isolates), and BSIs (5 isolates) was assessed by analysing REP-PCR DNA band patterns with more than 70% similarity 38  www.nature.com/scientificreports/related E. coli strains from different diseases and non-genetically related E. coli strains from the same disease, which contained the highest numbers of VGs and ARGs, were selected for WGS.A total of 12 E. coli strains, including 4 from pyometra, 5 from PAs, and 3 from BSIs, were chosen.The genomes of seven characterised 3GC R E. coli strains associated with global high-risk clones from canine and feline UTIs 12 , including ST38, ST131, ST617, ST641, ST648, and ST1193, were additionally sequenced for comparison.Genomic DNA extraction was performed using the G-spin™ Total DNA Extraction Mini Kit (intron Bio, South Korea) for short-read Illumina sequencing and the Nucleospin® Tissue DNA extraction kits (Machery-Nagel, Germany) for long-read Oxford Nanopore Technologies (ONT) sequencing.DNA libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs, USA) and were subsequently loaded into an Illumina NextSeq system (Illumina, USA) to obtain 150 bp pair-end reads by a private service company (Celemic, South Korea).For longread sequencing, the Native Barcoding Kit (SQK-NBD112.24)was utilised for multiplex library preparation from the genomic DNA, followed by loading into an R10.4 flowcell (FLO-MIN112) (ONT, UK).Trimmomatic v.0.39 was employed to trim short reads and remove adaptors to obtain high-quality reads 39 .Guppy v.6.3.8 was used for base-calling and de-multiplexing of long reads.Unicycler pipeline v.0.4.8 was employed for the assembly of short and long reads, resulting in generation of circular chromosomes and plasmids 40 .

Pan-genome, phylogenetic and bioinformatic analyses
The pan-genome of 19 sequenced strains was analysed to identify core genes and accessory genes using Roary version 3.11.2 41.The coreSNPs were extracted from core genome alignment using SNP sites for constructing a phylogenetic tree using RAxML version 8 with 1000 bootstraps 42,43 .The coreSNP tree was visualised using Interactive Tree of Life (iTOL) (https:// itol.embl.de/ itol.cgi).STs and complex types were determined based on Achtman's MLST and cgMLST, respectively, by submitting short reads to EnteroBase (enterobase.warwick.ac.uk) 44 .A minimum spanning tree based on cgMLST of 2513 core genes was generated using GrapeTree (available at enterobase.warwick.ac.uk) to illustrate genomic relationships among the whole-genome-sequenced strains 45 .Acquired ARGs and point mutations of QRDR associated with FQ R were detected using ResFinder v.4.1 and the NCBI AMR Finder tool 46,47 .VGs were identified using the Virulence Factor Database (VFDB) 48 .MGEs, including plasmids and PAIs, were identified using PlasmidFinder and IslandViewer 4, respectively 49,50 .The complete genome of UPEC strain CFT073 was utilised as the reference genome for PAI analysis.FimHTyper 1.0 and SerotypeFinder 2.0 were employed to determine E. coli FimH types and serotypes, respectively 51,52 .The sequenced contigs were submitted to the NCBI Prokaryotic Genome Annotation Pipeline for ORF prediction and gene annotation.

Statistical analysis
Fisher's exact test was used to ascertain the association between various phylogroups and/or disease patterns and the presence of the VGs, and ExPEC and/or UPEC genotypes and disease patterns.The investigation was assessed by using IBM ® SPSS ® Statistics version 26.Statistical significance was set at p < 0.05.

Figure 1 .
Figure 1.Minimum spanning tree based on core genome multilocus sequence typing (cgMLST) of 19 Escherichia coli strains.The isolates from pyometra (4 isolates, pink circles), prostatic abscesses (5 isolates, blue circles), bloodstream infections (3 isolates, red circles) and urinary tract infections (7 isolates, yellow circles) in dogs and cats are indicated in individual circles, presenting E. coli strains, sequence types (ST), and O:H antigens.Black lines and numbers indicate genetic relatedness and allele differences, respectively.

Figure 3 .
Figure 3. Pathogenicity islands (PAIs) identified in 19 Escherichia coli strains.PAIs containing virulence genes inserted at the insertion sites of 19 Escherichia coli strains isolated from canine pyometra (4 isolates) and prostatic abscesses (5 isolates), and bloodstream infections (3 isolates) and urinary tract infections (7 isolates) in dogs and cats, classified into phylogroups and sequence types (ST).Red squares indicate the presence of virulence genes in each PAI.

Table 1 .
Virulence genes associated with extraintestinal infections in Escherichia coli classified into phylogroups and sources.
35ylogroups and ExPEC VG carriage of the 104 E. coli were identified using PCR-based methods.Genomic DNA was extracted using Nucleospin ® Tissue DNA extraction kits (Machery-Nagel, Germany).Clermont E. coli phylotyping, which consists of a quadruplex PCR panel and two simplex PCRs, was performed to differentiate E. coli phylogroup A, B1, B2, C, D, E, F and E. coli cryptic clades35.VGs associated with extraintestinal pathogenesis of E.